Human Cell Extracts Research Articles

Ubiquitin ligase ARF-BP1/Mule modulates base excision repair

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady-state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF-BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase beta (Pol beta), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol beta and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol beta and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins.

MCM10 mediates RECQ4 association with MCM2-7 helicase complex during DNA replication

Mutations in RECQ4, a member of the RecQ family of DNA helicases, have been linked to the progeroid disease Rothmund-Thomson Syndrome. Attempts to understand the complex phenotypes observed in recq4-deficient cells suggest a potential involvement in DNA repair and replication, yet the molecular basis of the function of RECQ4 in these processes remains unknown. Here, we report the identification of a highly purified chromatin-bound RECQ4 complex from human cell extracts. We found that essential replisome factors MCM10, MCM2-7 helicase, CDC45 and GINS are the primary interaction partner proteins of human RECQ4. Importantly, complex formation and the association of RECQ4 with the replication origin are cell-cycle regulated. Furthermore, we show that MCM10 is essential for the integrity of the RECQ4-MCM replicative helicase complex. MCM10 interacts directly with RECQ4 and regulates its DNA unwinding activity, and that this interaction may be modulated by cyclin-dependent kinase phosphorylation. Thus, these studies show that RECQ4 is an integral component of the MCM replicative helicase complex participating in DNA replication in human cells.

SLA/LP/tRNP (Ser)Sec antigen in autoimmune hepatitis: Identification of the native protein in human hepatic cell extract

A diagnostic subgroup of AIH type 1 is characterized by specific serum antibodies against soluble liver protein. The respective autoantigen was named SLA/LP/tRNP (Ser)Sec, after three homologous recombinant polypeptides were isolated from expression gene libraries. We analyzed human cultured liver cells for the human homologue of recombinant SLA/LP/tRNP (Ser)Sec by antigen purification. In addition, a monoclonal antibody was generated against recombinant SLA-p35, a truncated recombinant SLA-reactive polypeptide. With a positive patient serum, immune affinity chromatography was performed on the 52 kD-SLA main antigenic determinant pre-enriched by ion exchange chromatography. By mass spectrometry, the 52 kD-SLA/LP/tRNP (Ser)Sec autoantigen was unambiguously identified in the purification product. The identity of the recombinant SLA-p35 and its human homologue was further confirmed by a specific signal of the anti SLA-p35 monoclonal antibody with purified human SLA/LP/tRNP (Ser)Sec. The 48 kD-SLA species frequently comigrating in SLA-immunoblotting however was not identified by either approach. We conclude that the native counterpart of recombinant tRNP (Ser)(Sec) indeed is detectable with a molecular weight of 52 kD in soluble liver extract of human cells as the major antigenic component of SLA/LP/tRNP (Ser)Sec.

Application of Screening Methods, Shape Signatures and Engineered Biosensors in Early Drug Discovery Process

In this study, two unreported estrogen antagonists were identified using a combination of computational screening and a simple bacterial estrogen sensor. Molecules here presented were initially part of a group obtained from a library of over a half million chemical compounds, using the Shape Signatures method. The structures within this group were then clustered and compared to known antagonists based on their physico-chemical parameters, and possible binding modes of the compounds to the Estrogen Receptor alpha (ER alpha) were analyzed. Finally, thirteen candidate compounds were purchased, and two of them were shown to behave as potential subtype-selective estrogen antagonists using a set of bacterial estrogen biosensors, which included sensors for ER alpha, ER beta, and a negative control thyroid hormone beta biosensor. These activities were then analyzed using an ELISA assay against activated ER alpha in human MCF-7 cell extract. Two new estrogen receptor antagonists were detected using in silico Shape Signatures method with an engineered subtype-selective bacterial estrogen biosensor and commercially available ELISA assay. Additional thyroid biosensor control experiments confirmed no compounds interacted with human thyroid receptor beta. This work demonstrates an effective combination of computational analysis and simple bacterial screens for rapid identification of potential hormone-like therapeutics.

Incision-dependent and error-free repair of (CAG)n/(CTG)n hairpins in human cell extracts

Expansion of CAG/CTG trinucleotide repeats is associated with certain familial neurological disorders, including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism contributes to their expansion. However, the molecular mechanism(s) by which cells remove CAG/CTG hairpins remain unknown. Here, we demonstrate that human cell extracts can catalyze error-free repair of CAG/CTG hairpins in a nick-directed manner. The repair system specifically targets CAG/CTG tracts for incisions in the nicked DNA strand, followed by DNA resynthesis using the continuous strand as a template, thereby ensuring CAG/CTG stability. PCNA is required for the incision step of the hairpin removal, which utilizes distinct endonuclease activities for individual CAG/CTG hairpins depending on their strand locations and/or secondary structures. The implication of these data for understanding the etiology of neurological diseases and trinucleotide repeat instability is discussed.

Biodegradable Protections for Nucleoside 5′-Monophosphates: Comparative Study on the Removal of O-Acetyl and O-Acetyloxymethyl Protected 3-Hydroxy-2,2-bis(ethoxycarbonyl)propyl Groups

The applicability of 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl and 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl groups as biodegradable phosphate protecting groups for nucleoside 5'-monophosphates has been studied in a HEPES buffer at pH 7.5. Enzymatic deacetylation with porcine carboxyesterase triggers the removal of the resulting 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl and 3-hydroxymethoxy-2,2-bis(ethoxycarbonyl)propyl groups by retro-aldol condensation and consecutive half acetal hydrolysis and retro-aldol condensation, respectively. The kinetics of these multistep deprotection reactions have been followed by HPLC, using appropriately protected thymidine 5'-monophosphates as model compounds. The enzymatic deacetylation of the 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl 5'-triester (2) is 25-fold faster than the deacetylation of its 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl-protected counterpart 1, and the difference in the deacetylation rates of the resulting diesters, 12b and 12a, is even greater. With 2, conversion to thymidine 5'-monophosphate (5'-TMP) is quantitative, while conversion of 1 to 5'-TMP is accompanied by formation of thymidine. Consistent with the preceding observations, quantitative release of 5'-TMP from 2 has been shown to take place in a whole cell extract of human prostate cancer cells.

Identification of uranyl binding proteins from human kidney-2 cell extracts by immobilized uranyl affinity chromatography and mass spectrometry

To improve our knowledge on protein targets of uranyl ion (UO 2 2+), we set up a proteomic strategy based on immobilized metal-affinity chromatography (IMAC). The successful enrichment of UO 2 2+-interacting proteins from human kidney-2 (HK-2) soluble cell extracts was obtained using an ion-exchange chromatography followed by a dedicated IMAC process previously described and designed for the uranyl ion. By mass spectrometry analysis we identified 64 proteins displaying varied functions. The use of a computational screening algorithm along with the particular ligand-based properties of the UO 2 2+ ion allowed the analysis and categorization of the protein collection. This profitable approach demonstrated that most of these proteins fulfill criteria which could rationalize their binding to the UO 2 2+-loaded phase. The obtained results enable us to focus on some targets for more in-depth studies and open new insights on its toxicity mechanisms at molecular level.

High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes

Thirty years of research on gene transcription has uncovered a myriad of factors that regulate, directly or indirectly, the activity of RNA polymerase II (RNAPII) during mRNA synthesis. Yet many regulatory factors remain to be discovered. Using protein affinity purification coupled to mass spectrometry (AP-MS), we recently unraveled a high-density interaction network formed by RNAPII and its accessory factors from the soluble fraction of human cell extracts. Validation of the dataset using a machine learning approach trained to minimize the rate of false positives and false negatives yielded a high-confidence dataset and uncovered novel interactors that regulate the RNAPII transcription machinery, including a new protein assembly we named the RNAPII-Associated Protein 3 (RPAP3) complex.

Photoactivated CdTe/CdSe Quantum Dots as a Near Infrared Fluorescent Probe for Detecting Biothiols in Biological Fluids

The important roles of biothiols in biological systems have attracted great interest in the determination of biothiols. Although great progress has been made in fluorescent biothiol probes, near-infrared (NIR) fluorescent probes for biothiols are rather few even such NIR probes can avoid interference from biological media such as tissue autofluorescence and scattering light, and thereby facilitate relatively interference-free sensing. Herein, we report photoactivated CdTe/CdSe quantum dots (QDs) as a novel NIR fluorescent probe for biothiols. The photoactivated CdTe/CdSe QDs based NIR fluorescent probe offers good sensitivity and selectivity for detecting cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) in the presence of 20 other amino acids, main relevant metal ions, and some other molecules in biological fluids. The recovery of spiked 5.0 microM thiols in human urine, plasma, and cell extracts ranges from 90% to 109%. The precision for nine replicate measurements of the thiols at 5.0 microM is in the range from 1.6% to 1.8%. The detection limits for Cys, Hcy, and GSH are 131, 26, and 20 nM, respectively. This assay is based on both the superior photoactivity of CdTe/CdSe QDs and the strong affinity of thiols to photoactivated CdTe/CdSe QDs. The addition of thiols into the photoactivated CdTe/CdSe QDs improves the passivation of the illumination-induced traps, meanwhile reduces most of Se(IV) and Te(IV) on the surface of photoactivated CdTe/CdSe QDs so as to improve the fluorescence property.

Probing the Functional Impact of Sequence Variation on p53-DNA Interactions Using a Novel Microsphere Assay for Protein-DNA Binding with Human Cell Extracts

The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs). Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation—including polymorphisms—and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD) for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt) variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks.

Enhancing of GM3 synthase expression during differentiation of human blood monocytes into macrophages as in vitro model of GM3 accumulation in atherosclerotic lesion

In previous studies, we showed that ganglioside levels (GM3 being the main ganglioside) in human aortic intima isolated from atherosclerotic lesions were 5 times greater compared to intima from non-diseased vascular areas. Recently, we found that GM3 and GM3 synthase levels in differentiated in vitro macrophages were five and ten times higher, respectively, compared to freshly isolated human monocytes. In this article, we report that GM3 synthase mRNA levels were significantly higher in differentiated human monocyte-derived macrophages compared to monocytes and in atherosclerotic aorta compared to normal aorta. The depletion of GM3 synthesis in cultured monocyte-derived macrophages with DL-threo-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, an inhibitor of ganglioside synthesis, delayed the acquisition of CD206 antigen, prevented the loss of CD163 antigen and enhanced anti-inflammatory cytokine (CCL18) secretion. In the current study, we performed purification of CMP-N-acetylneuraminic acid:lactosylceramide alpha2,3-sialyltransferase (GM3 synthase) from Triton X-100 extract of human blood mononuclear cells by immunoaffinity chromatography on Sepharose coupled with anti-GM3 synthase antibody. Comparison with several glycolipid substrates showed high specificity of the purified enzyme for lactosylceramide. The apparent K(M) for lactosylceramide and CMP-NeuAc were 101 and 180 muM, respectively. Analysis of the purified enzyme by SDS-PAGE followed by the anti-GM3 synthase antibody probing detected two bands with apparent molecular masses of 60 and 64 kDa. There were no other protein bands as revealed by Coomassie Blue staining. Thus, ganglioside GM3 may be considered as a physiological modulator of macrophage differentiation in human atherosclerotic aorta. The presented data suggest that up-regulation of GM3 levels is an element of monocyte/macrophage differentiation that provides a tool for control of macrophage accumulation in inflammatory loci.

Interference of mismatch and base excision repair during the processing of adjacent U/G mispairs may play a key role in somatic hypermutation

In eukaryotic mismatch repair (MMR), degradation of the error-containing strand initiates at nicks or gaps that can be up to a kilobase away from the mispair. These discontinuities may be the ends of Okazaki fragments or the 3'-termini of the leading strands during replication, whereas the termini of invading strands may fulfill this role during recombination. Here we show that, in extracts of human cells, MMR can initiate also at sites of ongoing base excision repair. Although unlikely under normal circumstances, this situation may arise in vivo during somatic hypermutation (SHM) and class switch recombination of Ig genes, where activation-induced cytidine deaminase (AID) generates multiple U/G mismatches in the variable or switch regions. Uracil should normally be excised by base excision repair (BER), but we show here that MMR proteins activated by a nearby mismatch interfere with uracil processing to generate long single-stranded gaps. We postulate that, in a subset of the repair events, filling-in of the MMR-generated gaps might be catalyzed by the error-prone polymerase-eta, rather than by the high-fidelity polymerase-delta. Because polymerase-eta has a propensity to misinsertions opposite adenine residues, the above mechanism would help explain why SHM affects not only C/G, but also A/T base pairs.

Identification of target antigens of self‐reactive IgG in intravenous immunoglobulin preparations

Intravenous immunoglobulin (IVIg) contains a wide range of self-reactive immunoglobulins (Ig) G. Acidic pH is known to increase the reactivity of purified IgG with self-antigens. We describe here the target antigens of IgG autoantibodies in IVIg and analyze the influence of acidic pH on IgG reactivities. We used 2-DE and immunoblotting with protein extracts of human umbilical vein endothelial cells (HUVEC) and HEp-2 cells. Two IVIg preparations obtained by ethanol fractionation [one with an acidic pH step (acidic-IVIg) and one with beta-propiolactone (propiolactone-IVIg)] and a pool of sera from 12 healthy individuals were tested. Serum IgG of 3 healthy individuals and IgG purified from the same sera with elution at pH 2.8 were also tested individually. Finally, propiolactone-IVIg was acidified at pH 2.8. IgG obtained with a step at low pH recognized many more target spots than IgG obtained without acidic pH. Our findings demonstrate that an acidic pH step artificially enlarges the repertoire of self-reactive IgG. Thus, protein spots recognized by IgG in propiolactone-IVIg represent the core set of self-antigens targeted by IVIg. Overall, 96 proteins were identified by MS. Fourteen were recognized in both extracts including glycolysis proteins such as alpha-enolase, RNA processing and cytoskeletal proteins such as lamin-A/C.

Angiotensin II Stimulated Angiogenesis is Dependent on p38 MAPK and is Inhibited by Statins

Recent data suggest that angiogenesis plays a role in the development of atherosclerotic plaques. Previous studies have demonstrated that Angiotensin II (AII) induces angiogenesis in various systems, but its mechanism is not well understood. Our study tries to determine the molecular mechanism of the AII stimulated angiogenesis and the relationship between lipid metabolism and AII signaling. Here we show that the HMG‐CoA reductase inhibitor, Simvastatin, interferes with AII stimulated angiogenesis via the inhibition of p38 MAPK activation. Western blots using extracts of human umbilical cord vein endothelial cells (HUVEC) treated with AII demonstrated a time‐dependent increase in the phosphorylation of p38 (2.8±0.3 fold at 10 min and 6.0±0.2 fold at 20 min). Pretreatment of HUVEC with Simvastatin dose‐dependently inhibited AII stimulated p38 phosphorylation (62±19% reduction at 0.01 ?M and 83±17% reduction at 1 ?M). AII stimulated angiogenesis by 1.6±0.3 fold in a HUVEC Matrigel tube formation assay. Simvastatin treatment showed a dose‐dependent inhibition of AII stimulated tube formation (60% inhibition at 0.1 ?M and complete inhibition at 1 ?M). p38 inhibitor, SB203580, inhibited AII stimulated tube formation 98±18%. Our results suggest that AII induces angiogenesis, at least in part, via activation of p38, and that statins interfere with AII stimulated angiogenesis via the inhibition of this pathway.

A Simulated MS/MS Library for Spectrum-to-spectrum Searching in Large Scale Identification of Proteins

Identifying peptides from mass spectrometric fragmentation data (MS/MS spectra) using search strategies that map protein sequences to spectra is computationally expensive. An alternative strategy uses direct spectrum-to-spectrum matching against a reference library of previously observed MS/MS that has the advantage of evaluating matches using fragment ion intensities and other ion types than the simple set normally used. However, this approach is limited by the small sizes of the available peptide MS/MS libraries and the inability to evaluate the rate of false assignments. In this study, we observed good performance of simulated spectra generated by the kinetic model implemented in MassAnalyzer (Zhang, Z. (2004) Prediction of low-energy collision-induced dissociation spectra of peptides. Anal. Chem. 76, 3908-3922; Zhang, Z. (2005) Prediction of low-energy collision-induced dissociation spectra of peptides with three or more charges. Anal. Chem. 77, 6364-6373) as a substitute for the reference libraries used by the spectrum-to-spectrum search programs X!Hunter and BiblioSpec and similar results in comparison with the spectrum-to-sequence program Mascot. We also demonstrate the use of simulated spectra for searching against decoy sequences to estimate false discovery rates. Although we found lower score discrimination with spectrum-to-spectrum searches than with Mascot, particularly for higher charge forms, comparable peptide assignments with low false discovery rate were achieved by examining consensus between X!Hunter and Mascot, filtering results by mass accuracy, and ignoring score thresholds. Protein identification results are comparable to those achieved when evaluating consensus between Sequest and Mascot. Run times with large scale data sets using X!Hunter with the simulated spectral library are 7 times faster than Mascot and 80 times faster than Sequest with the human International Protein Index (IPI) database. We conclude that simulated spectral libraries greatly expand the search space available for spectrum-to-spectrum searching while enabling principled analyses and that the approach can be used in consensus strategies for large scale studies while reducing search times.

Expression of reverse cholesterol transport proteins ATP-binding cassette A1 (ABCA1) and scavenger receptor BI (SR-BI) in the retina and retinal pigment epithelium

Aims:Excessive lipid accumulation in Bruch’s membrane (BrM) is a hallmark of ageing, the major risk factor for age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells may utilise reverse cholesterol...

Replication protein A modulates the activity of human telomerase in vitro

Our aim was to investigate how replication protein A (RPA) in a wide range of concentration can regulate the activity of human telomerase. We used an in vitro system based on human cell extracts with or without RPA. It has been shown that removal of RPA leads to loss of telomerase activity and addition of RPA restores telomerase activity and at the same time promotes telomerase processivity. However, high excess of RPA inhibited telomerase processivity and caused the synthesis of relatively short DNA fragments (about 50-100 nucleotides). We assume that, together with other telomere-binding proteins, RPA may take part in activation of telomere overhang elongation by telomerase at a certain stage of a cell cycle as well as in regulation of telomere length.

Repair of DNA damage induced by anthanthrene, a polycyclic aromatic hydrocarbon (PAH) without bay or fjord regions

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants, formed during incomplete burning of coal, oil and gas. Several PAHs have carcinogenic and mutagenic potencies, but these compounds must be activated in order to exert their mutagenic effects. One of the principal pathways proposed for metabolic activation of PAHs involves the cytochrome P450 enzymes. The DNA damaging potential of cytochrome P450-activated PAHs is generally associated with their bay and fjord regions, and the DNA repair response of PAHs containing such regions has been thoroughly studied. However, little is known about the repair of DNA damage resulting from metabolites from PAHs without bay and fjord regions. We have investigated the six-ringed PAH anthanthrene (dibenzo[ def, mno]chrysene), which does not posses bay or fjord motifs. We analyzed the repair profile of human cell extracts and of cell cultures in response to DNA damage induced by cytochrome P450-activated anthanthrene. In cell extracts, functional nucleotide excision repair (NER) and mismatch repair (MMR) activities were necessary to trigger a response to anthanthrene metabolite-induced DNA damage. In cell cultures, NER was responsible for the repair of anthanthrene metabolite-induced DNA damage. However, when the NER pathway was inactivated, a residual repair pathway performed the DNA repair.

Physical and Functional Interaction between Human Oxidized Base-specific DNA Glycosylase NEIL1 and Flap Endonuclease 1

The S phase-specific activation of NEIL1 and not of the other DNA glycosylases responsible for repairing oxidatively damaged bases in mammalian genomes and the activation of NEIL1 by proliferating cell nuclear antigen (PCNA) suggested preferential action by NEIL1 in oxidized base repair during DNA replication. Here we show that NEIL1 interacts with flap endonuclease 1 (FEN-1), an essential component of the DNA replication. FEN-1 is present in the NEIL1 immunocomplex isolated from human cell extracts, and the two proteins colocalize in the nucleus. FEN-1 stimulates the activity of NEIL1 in vitro in excising 5-hydroxyuracil from duplex, bubble, forked, and single-stranded DNA substrates by up to 5-fold. The disordered region near the C terminus of NEIL1, which is dispensable for activity, is necessary and sufficient for high affinity binding to FEN-1 (K(D) approximately = 0.2 microm). The interacting interface of FEN-1 is localized in its disordered C-terminal region uniquely present in mammalian orthologs. Fine structure mapping identified several Lys and Arg residues in this region that form salt bridges with Asp and Glu residues in NEIL1. NEIL1 was previously shown to initiate single nucleotide excision repair, which does not require FEN-1 or PCNA. The present study shows that NEIL1 could also participate in strand displacement repair synthesis (long patch repair (LP-BER)) mediated by FEN-1 and stimulated by PCNA. Interaction between NEIL1 and FEN-1 is essential for efficient NEIL1-initiated LP-BER. These studies strongly implicate NEIL1 in a distinct subpathway of LP-BER in replicating genomes.

342 POSTER 2-[18F] fluoro-2-deoxy-d-glucose positron emission tomography is an early biomarker for tumor growth inhibition of human Colo205 xenografts by the novel and selective CENP-E inhibitor, GSK923295A

Recent evidence suggests that human O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that protects the genome against mutagens and accords tumor resistance to many anticancer alkylating agents, may have other roles besides repair. Therefore, we isolated MGMT-interacting proteins from extracts of HT29 human colon cancer cells using affinity chromatography on MGMT–Sepharose. Specific proteins bound to this column were identified by electrospray ionization tandem mass spectrometry and/or Western blotting. These procedures identified >60 MGMT-interacting proteins with diverse functions including those involved in DNA replication and repair (MCM2, PCNA, ORC1, DNA polymerase δ, MSH-2, and DNA-dependent protein kinase), cell cycle progression (CDK1, cyclin B, CDK2, CDC7, CDC10, 14-3-3 protein, and p21waf1/cip1), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1α), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90α and β, also bound strongly with MGMT. The DNA repair activity of MGMT was greatly enhanced in the presence of interacting proteins or histones. These data, for the first time, suggest that human MGMT is likely to have additional functions, possibly, in sensing and integrating the DNA damage/repair-related signals with replication, cell cycle progression, and genomic stability.